The present invention relates to antimicrobial peptides which are resistant to proteases and which have the ability to reduce the extent of protease degradation of peptides, polypeptides and proteins in plants.
Antimicrobial peptides are produced by a wide range of organisms as part of their defense against infection. See Hancock & Lehrer, 1998, TIBTECH, 16:82–88; Everett, 1994, Chpt. 20 In: Natural and Engineered Pest Management Agents, eds. Hedin, Menn & Hollingworth, ACS Symposium Series 551, pp. 278–91. Examples of such peptides include cecropins, attacins and diptericins which are involved in cell-free immunity in insects, the apidaecins from honeybees, the defensins from mammalian phagocytes, and the magainins from frog skin. Plants also produce certain classes of antimicrobial peptides which are thought to play a role in resistance to microbial plant pathogens. See Broekaert et al., 1997, Critical Reviews in Plant Sciences, 16:297–323.
Plants have been genetically engineered to produce antimicrobial peptides, both natural and synthetic to increase resistance to disease. See Jaynes et al., 1987, BioEssays, 6:263-70; Hancock and Lehrer, 1998, TIBTECH, 16:82–88. Unfortunately, this approach has met with very limited success. Either the amount of peptide produced by the transgenic plant is too small and/or the plants are no less Lehrer, 1998, TIBTECH, 16:82–88. A major limitation to the expression of foreign peptides in transgenic plants is due to the susceptibility of the foreign peptides to rapid degradation by proteases. For example, transgenic potato cultivars which express a gene encoding the antimicrobial peptide cecropin B at levels up to 0.6% of total mRNA produce no detectable cecropin B peptide and no improvement in resistance to potato soft rot. See Sjefke et al., 1995, American Potato Journal, 72:437–45. Similar studies in tobacco have demonstrated that expression of cecropin B genes also does not result in detectable cecropin B peptide levels and resistance to bacterial infections. See Florack et al., 1995, Transgenic Research 4:132–41. Studies have also shown that cecropin B and antimicrobial peptides related to magainins are rapidly degraded by proteases in the intercellular fluid of plant leaves. See Mills et al., 1994, Plant Science, 104:17–22 and Everett, 1994, Chpt. 20 In: Natural and Engineered Pest Management Agents, eds. Hedin, Menn & Hollingworth, ACS Symposium Series 551, pp. 278–91.
One proposed solution to the problem of peptide instability due to protease degradation has been to identify the protease-sensitive sites within a particular peptide and to design amino acid substitutions that increase the stability of peptides to plant proteases while retaining the antimicrobial activity of the peptides. This approach resulted in a synthetic magainin derivative having the amino acid sequence Met-Gly-Ile-Gly-Lys-Phe-Leu-Arg-Glu-Ala-Gly-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Lys-Pro (SEQ ID NO:1) that had enhanced stability against proteases found in the intercellular fluid of plant tissues and was therefore an improved candidate for use in or on plants. See Everett, 1994, Chpt. 20 In: Natural and Engineered Pest Management Agents, eds. Hedin, Menn & Hollingworth, ACS Symposium Series 551, pp. 278–91; U.S. Pat. Nos. 5,424,395 and 5,519,115.
Another proposed solution to the problem of peptide instability has been to produce the reverse- or retro-analogs of natural antimicrobial peptides or their synthetic derivatives. See U.S. Pat. No. 5,519,115, and Merrifield et al., 1995, PNAS, 92:3449–53. Such reverse-peptides retain the same general three-dimensional structure (e.g., alpha-helix) as the parent peptide except for the conformation around internal protease-sensitive sites and the characteristics of the N- and C-termini. Reverse peptides are purported not only to retain the biological activity of the non-reversed “normal” peptide but may possess enhanced properties, including increased antibacterial activity and reduced hemolysis. See Iwahori et al., 1997, Biol. Pharm. Bull. 20:267–70.
Indolicidin, having the amino acid sequence Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg, (SEQ ID NO:2) is a potent antimicrobial tridecapeptide. It was originally purified from cytoplasmic granules of bovine neutrophils. See Selsted et al., 1993, J. Biol. Chem., 267:4292–95. It is a member of a class of proline-rich peptides that have been recovered from the leukocytes of different mammals, a marine invertebrate and insect haemolymph. See Hancock and Lehrer, 1998, TIBTECH, 16:82–88. The antimicrobial potencies of natural and synthetic indolicidin are identical. See Van Abel et al., 1995, Int. J. Protein Res. 45:401–09. The mode of antibacterial action of indolicidin has been reported to be based on the disruption of the cytoplasmic membrane by channel formation. See Falla et al., 1996, J. Biol. Chem. 32:19298–303. More recently, it has been suggested that membrane permeabilization is likely to occur due to deformation of the membrane surface rather than formation of transmembrane channels by indolicidin and its analogs. See Subbalakshmi et al., 1998, J. Biosci., 23:9–13.
Numerous analogs of indolicidin have been synthesized and tested in attempts to evaluate the requirements for antimicrobial and hemolytic activities, and to increase activity. Subbalakshmi et al. (FEBS Letters 395:48–52 (1996)) reports that peptides in which proline was replaced by alanine and tryptophan was replaced by phenylalanine exhibit antibacterial activities comparable to that of indolicidin. The replacement of tryptophan by phenylalanine, however resulted in a loss of hemolytic activity. Falla and Hancock (Antimicrobial Agents and Chemotherapy, 41:771–75 (1997)) tested a synthetic peptide, CP-11, Ile-Leu-Lys-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg, (SEQ ID NO:3) based on indolicidin, which was designed to increase the number of positively charged residues, maintain the short length (13 amino acids), and enhance the amphipathicity relative to indolicidin. They found that CP-11 had better activity against E. coli, Pseudomonas aeruginosa, and Candida albicans, but reduced activity against Staphylococcus aureus. Lim et al. (J. Biochem. Mol. Biol. 30:229–33 (1997)) tested the effects of substituting certain tryptophan, proline or arginine residues in indolicidin. Substitutions of some tryptophan residues by isoleucine or glycine were tolerated but substitution of Pro7 with alanine significantly reduced activity against E. coli. Substitutions of either Arg12 or Arg13 with alanine also reduced biological activity.